Project Summary Vaccination is one of the greatest inventions in human medicine history. The eradication of smallpox was announced in 1979 and this success inspired continuing efforts in developing vaccines against other devastating diseases, such as HIV, cancer, and influenza pandemics (1-4). Unfortunately, the conventional vaccine design relying on antibody responses has been shown to be inadequate in these cases, suggesting that it is also critical to activate the other arm of the human adaptive immune system - T cell responses (5-7). Unlike antibody responses that could be routinely monitored by doctors, there is no defined standard for them to tell how good a T cell response is (5, 8-10). Therefore, the definition of such a standard could greatly improve our ability to predict vaccine effectiveness and help set new standards for immune monitoring. One major impediment to defining a healthy T cell response is that there is no high throughput method available to measure how many different T cells (each recognizes a specific pathogenic peptide) are activated, in other words, the breadth of the T cell response. The current best method for measuring different T cells simultaneously uses a mixture of fluorescent protein complexes (tetramers), each of which specifically binds one type of T cell (11). Due to the intrinsic limitations of the fluorescent dyes, it is very difficult to measure more than 15 different T cells simultaneously (12, 13). In addition, this method also showed limited sensitivity - T cells with less than 0.01% abundance (~ a few dozens of T cells) could not be reliably detected. Therefore, the goal of this proposal is to develop a highly multiplexed and sensitive method to profile the T cell repertoire. Specifically, we will tag the protein complex that binds a specific T cell with a unique DNA molecule. As a result, each T cell will be barcoded with a unique DNA sequence, which can then be detected using a DNA chip. The use of DNA as barcode offers us the potential to improve the multiplexed detection capacity of different T cells by several orders of magnitude, thanks to the exquisite specificity of DNA hybridization and high throughput data processing of DNA chips (millions of different DNA molecules can be analyzed in parallel using a single chip). In addition, the detection sensitivity could be significantly improved by including a DNA amplification step (one billion DNA molecules can be obtained from just a single copy within hours) before chip analysis. The successful development of the proposed method will enable us to detect as many as tens of thousands of different T cells simultaneously, and as few as just one T cell.